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Tumor-infiltrating B lymphocytes as an efficient source of highly specific immunoglobulins recognizing tumor cells.

Pavoni E, Monteriù G, Santapaola D, Petronzelli F, Anastasi AM, Pelliccia A, D'Alessio V, De Santis R, Minenkova O - BMC Biotechnol. (2007)

Bottom Line: In the present study, we analyzed the significance of local humoral response provided by tumor-infiltrating lymphocytes in breast cancer patients.Reactivity of novel scFvs was confirmed by ELISA, immunohistochemistry, fluorescence staining and flow cytometry.The described method is very promising for development of antitumor antibodies, potentially useful for diagnostic and therapeutic approaches.

View Article: PubMed Central - HTML - PubMed

Affiliation: Kenton Srl, c/o Sigma-Tau SpA, via Pontina, km 30,400, 00040 Pomezia (RM), Italy. pavoni@kenton.it

ABSTRACT

Background: There is much evidence that tumor cells elicit a humoral immune response in patients. In most cases, the presence of antibodies in peripheral blood is detected only in small proportion of patients with tumors overexpressing the corresponding antigen. In the present study, we analyzed the significance of local humoral response provided by tumor-infiltrating lymphocytes in breast cancer patients.

Methods: The ability of a patient's immune system to produce specific antibodies inside tumor tissue, capable of recognizing tumor cells, was explored through analysis of the oligoclonality of antibodies derived from tumor-infiltrating lymphocytes and construction of a series of recombinant antibody libraries in scFv format, derived from breast tumor-infiltrating B lymphocytes. These libraries and one from peripheral blood lymphocytes of a single breast cancer patient were panned against three purified surface tumor antigens, such as CEA, MUC1 and ED-B domain, and against intact MCF7 breast carcinoma cells.

Results: Application of novel display vector, pKM19, allowed isolation of a large panel of breast cancer-specific antibodies against known tumor antigens, as well as against breast carcinoma cells. Reactivity of novel scFvs was confirmed by ELISA, immunohistochemistry, fluorescence staining and flow cytometry. We demonstrated that seven of ten primary breast tumor specimens, obtained using discarded surgical material, could be exploited as an appropriate source for generation of phage display libraries, giving highly specific antitumor antibodies which recognize heterologous tumor cells.

Conclusion: Local humoral immune response within tumor tissue in breast cancer patients frequently has an oligoclonal character. Efficient selection of specific antitumor antibodies from recombinant antibody libraries, derived from such oligoclonal tumor-infiltrated B lymphocytes, indicates the presence of natural immune response against tumor antigens in these patients. The described method is very promising for development of antitumor antibodies, potentially useful for diagnostic and therapeutic approaches.

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Related in: MedlinePlus

Origin of anti-MCF7 scFv antibodies. One μL of each scFv phage library was amplified by PCR using oligonucleotide primers specific for analyzed antibody genes. Corresponding PEG-purified phage was used as positive control (last line). The irrelevant anti-SP2 antibody gene of known origin, selected earlier from scFvEC23 library, was also tested.
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Figure 4: Origin of anti-MCF7 scFv antibodies. One μL of each scFv phage library was amplified by PCR using oligonucleotide primers specific for analyzed antibody genes. Corresponding PEG-purified phage was used as positive control (last line). The irrelevant anti-SP2 antibody gene of known origin, selected earlier from scFvEC23 library, was also tested.

Mentions: MIX7-MIX39 scFv antibodies were selected from a mixture of PBL and TIL-derived libraries. We investigated the origin of these antibodies to see which type of library functions better under equal selection conditions. One μL of each amplified library was used as template for PCR amplification with a pair of oligonucleotide primers specific for each antibody (Fig. 4). This analysis shows that five tested scFv antibodies, isolated from a mixture of libraries, belong to TIL-derived antibodies. Antibody genes of MIX7 and MIX25 (having the same heavy chain as MIX12), and MIX8 (similar to MIX39) are believed to have a similar origin. Regarding the irrelevant anti-SP2 antibody, selected earlier from an scFvEC23 library [35], its origin from a PBL-derived library was confirmed.


Tumor-infiltrating B lymphocytes as an efficient source of highly specific immunoglobulins recognizing tumor cells.

Pavoni E, Monteriù G, Santapaola D, Petronzelli F, Anastasi AM, Pelliccia A, D'Alessio V, De Santis R, Minenkova O - BMC Biotechnol. (2007)

Origin of anti-MCF7 scFv antibodies. One μL of each scFv phage library was amplified by PCR using oligonucleotide primers specific for analyzed antibody genes. Corresponding PEG-purified phage was used as positive control (last line). The irrelevant anti-SP2 antibody gene of known origin, selected earlier from scFvEC23 library, was also tested.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175506&req=5

Figure 4: Origin of anti-MCF7 scFv antibodies. One μL of each scFv phage library was amplified by PCR using oligonucleotide primers specific for analyzed antibody genes. Corresponding PEG-purified phage was used as positive control (last line). The irrelevant anti-SP2 antibody gene of known origin, selected earlier from scFvEC23 library, was also tested.
Mentions: MIX7-MIX39 scFv antibodies were selected from a mixture of PBL and TIL-derived libraries. We investigated the origin of these antibodies to see which type of library functions better under equal selection conditions. One μL of each amplified library was used as template for PCR amplification with a pair of oligonucleotide primers specific for each antibody (Fig. 4). This analysis shows that five tested scFv antibodies, isolated from a mixture of libraries, belong to TIL-derived antibodies. Antibody genes of MIX7 and MIX25 (having the same heavy chain as MIX12), and MIX8 (similar to MIX39) are believed to have a similar origin. Regarding the irrelevant anti-SP2 antibody, selected earlier from an scFvEC23 library [35], its origin from a PBL-derived library was confirmed.

Bottom Line: In the present study, we analyzed the significance of local humoral response provided by tumor-infiltrating lymphocytes in breast cancer patients.Reactivity of novel scFvs was confirmed by ELISA, immunohistochemistry, fluorescence staining and flow cytometry.The described method is very promising for development of antitumor antibodies, potentially useful for diagnostic and therapeutic approaches.

View Article: PubMed Central - HTML - PubMed

Affiliation: Kenton Srl, c/o Sigma-Tau SpA, via Pontina, km 30,400, 00040 Pomezia (RM), Italy. pavoni@kenton.it

ABSTRACT

Background: There is much evidence that tumor cells elicit a humoral immune response in patients. In most cases, the presence of antibodies in peripheral blood is detected only in small proportion of patients with tumors overexpressing the corresponding antigen. In the present study, we analyzed the significance of local humoral response provided by tumor-infiltrating lymphocytes in breast cancer patients.

Methods: The ability of a patient's immune system to produce specific antibodies inside tumor tissue, capable of recognizing tumor cells, was explored through analysis of the oligoclonality of antibodies derived from tumor-infiltrating lymphocytes and construction of a series of recombinant antibody libraries in scFv format, derived from breast tumor-infiltrating B lymphocytes. These libraries and one from peripheral blood lymphocytes of a single breast cancer patient were panned against three purified surface tumor antigens, such as CEA, MUC1 and ED-B domain, and against intact MCF7 breast carcinoma cells.

Results: Application of novel display vector, pKM19, allowed isolation of a large panel of breast cancer-specific antibodies against known tumor antigens, as well as against breast carcinoma cells. Reactivity of novel scFvs was confirmed by ELISA, immunohistochemistry, fluorescence staining and flow cytometry. We demonstrated that seven of ten primary breast tumor specimens, obtained using discarded surgical material, could be exploited as an appropriate source for generation of phage display libraries, giving highly specific antitumor antibodies which recognize heterologous tumor cells.

Conclusion: Local humoral immune response within tumor tissue in breast cancer patients frequently has an oligoclonal character. Efficient selection of specific antitumor antibodies from recombinant antibody libraries, derived from such oligoclonal tumor-infiltrated B lymphocytes, indicates the presence of natural immune response against tumor antigens in these patients. The described method is very promising for development of antitumor antibodies, potentially useful for diagnostic and therapeutic approaches.

Show MeSH
Related in: MedlinePlus